ConspectusThe clinical use of mRNA COVID-19 vaccines developed by Moderna and Pfizer-BioNTech has highlighted the critical role of ionizable lipid nanoparticles (LNPs) in the efficient loading, intracellular delivery, and cytoplasmic release of mRNAs. These LNPs typically comprise an ionizable lipid, a helper lipid, cholesterol, and a PEGylated-lipid, each contributing to the stability, structure, encapsulation efficiency, and nanoparticle-biology interactions of the final mRNA-LNPs bothand. Notably, the ionizable amino-lipids, ALC-0315 used in the BioNTech/Pfizer vaccine and SM-102 in the Moderna vaccine, possess similar molecular structures, featuring multiple saturated aliphatic chains linked to a tertiary amine group via ester bonds. The acidification-induced ionization behavior of these amino-lipids is essential for enabling endosomal escape and facilitating the intracellular transfection of therapeutic mRNAs. However, despite their widespread clinical use, the physicochemical property-biological interaction and function relationships for LNPs remain poorly understood, particularly regarding how the internal nanostructural evolution during endosomal maturation influences mRNA release, endosomal escape, and gene expression. The rudimentary understanding continues to impede the rational design and optimization of RNA therapeutics.With long-standing expertise in amphiphile self-assembly and structural characterization, especially inverse lyotropic liquid crystalline mesophase-forming lipids, our group seeks to address this critical knowledge gap by establishing a clear connection between pH-triggered mesophase transitions and the biological performance of mRNA-LNPs, with the aim of providing new mechanistic insight into how the internal nanostructure affects mRNA delivery efficiency. This Account focuses on the pH-dependent inverse mesostructural behavior of ionizable LNPs containing two COVID-19 mRNA vaccine ionizable lipids, ALC-0315 and SM-102. We have applied high-throughput and cutting-edge time-resolved synchrotron radiation small-angle X-ray scattering (SAXS) to investigate both static and kinetic self-assembly and structural transitions of these ionizable LNPs without and with nucleic acid cargos (including mRNAs, polyA tails, and plasmid DNAs) upon acidification. We further explored the influence of other components in LNPs, such as select structure-forming helper lipids (monoolein and phytantriol) and cholesterol, on their physicochemical properties, mesophase behavior, and gene delivery performance. Notably, we correlated the mesophase transition of LNPs, from nonordered state to ordered inverse micellar, hexagonal, and cubic phases, with their mRNA transfection efficiency in macrophage cells, providing mechanistic insight into the role of internal nanostructure in endosomal escape and gene expression. Moreover, we addressed the impact of protein coronas formed upon exposure to biological environments, which can significantly alter the LNP internal structure and delivery efficiency. Our findings suggest that protein corona-modulated phase behavior of LNPs may contribute to reported inconsistency betweenandperformance. Finally, we offer a perspective on future research trends in improving endosomal escape efficiency, promoting a passive nonendocytic cellular uptake pathway, modulating protein corona effects, monitoring the immune compatibility of PEG-free stabilizers, and leveraging of artificial intelligence approaches to accelerate formulation design and screening. Overall, this Account provides guidance for future mechanistic research with respect to LNP internal structures under various environmental and biological conditions, enabling the rational design of next-generation RNA therapeutics. in vitro in vivo in vitro in vivo
